A profound hemolytic anemia following exposure to arylamine drugs and environmental chemicals affects Americans of African and Mediterranean descent (Italian, Greek, etc.). In the 50's and 60's, extensive studies on primaquine and phenylhydrazine led to the concept that (re) active metabolites of the drugs damaged red cells by "oxidative stress". The hemotoxic metabolites however were not identified and the postulate of "oxidative stress" as the mechanism underlying drug-induced hemolytic anemia was not further developed. We have now identified hemolytic metabolites of aniline, dapsone and phenacetin and observed high hemolytic activity with a putative metabolite of primaquine. Mechanism studies using the hemolytic metabolite of depsone (DDS-NOH) in rat red cells indicated that DDS-NOH causes a), formation of disulfide-linked adducts between certain membrane skeletal proteins and hemoglobin monomers, and b), development of an extreme echinocyte morphology. These data, taken together with recent literature observations on acitive oxygen biochemistry, red cell skeletal protein function, and the normal mechanisms of splenic sequestration of "old" red cells, suggest an overall working hypothesis for the mechanism of drug-induced hemolytic anemia; viz that the toxic drug metabolites interact with oxyhemoglobin to generate oxygen free radicals, which in turn generate free radicals of hemoglobin sulfhydryl groups. Adduct formation between hemoglobin and membrane skeletal proteins results in loss of stuctural integrity and activation of senescent antibody binding sites on the cell surface, with subsequent macrophage phagocytosis. The objectives of the present studies are a), to indentify the hemolytic metobolites of primaquine, b) to define the role of acitve oxygen species in activating red cell protein sulfhydryl groups and in the commitment of DDS-NOH damaged red cells to splenic sequestration, c) to identify the domains of spectrin andd ankyrin to which the hemoglobin monomers bind and to assess the structural significance of this binding, and d), to determine whether or not the senescent antibody plays a role in the removal of DDS-NOH damaged red cells from the circulation. It is expected that these studies will determine the relative importance of N vs C oxidation products of primaquine in primaquine hemotoxicity, establish whether or not active oxygen species are obligatory in the hemolytic sequence, and provide a firm basis for more detailed resolution of how skeletal protein structure on the inside of the red cell membrane determines the properties and structures of the outer surface of the red cell.